Chronic hepatitis B virus infections affect about 300 million people worldwide and constitute a significant risk factor for fibrosis and hepatocellular carcinoma (HCC). HBV, a DNA virus, amplifies its genome via a pre-genomic RNA. This RNA is converted by HBV-encoded polymerase protein (pol), which functions as a reverse transcriptase, into DNA. The final product of this reaction is a partially double stranded DNA, which is converted to covalently closed circular DNA (cccDNA) in the nucleus cccDNA serves as a template for viral mRNA synthesis and proteins. cccDNA persists in the presence of the antivirals in current use, which act as chain terminators. We (Hostetler and colleagues) have previously shown that the acyclic nucleoside phosphonates, HPMPC and HPMPA are incorporated into viral DNA. Viral DNA templates containing these bases cannot be copied readily and have aberrations in DNA structure. In this study, we propose to investigate the incorporation of these agents into HBV DNA during the conversion from the RNA pregenome within the core particles and subsequent synthesis of cccDNA. In the R21 phase of the grant, we investigate the incorporation of radiolabeled HPMPA/HPMPC and its impact by analysis of viral DNA within core particles and finally the formation of cccDNA. Effect of these drugs on viral mRNA and protein synthesis will be analyzed to test the template activity of cccDNA containing HPMPA/HPMPC. In the second R33 phase, we propose to define the changes in duplex DNA containing these nucleotides. We will synthesize several templates containing HPMPA or HPMPC and assess the NMR solution structures of their duplex DNA versus control DNA duplexes. Aim 4 of R33 phase explores the impact of these drugs in the context of HBV infection using a cell line HepaRG that is widely used in the HBV field and recognized for its susceptibility to HBV infection. These studies will provide a unique opportunity to assess the potential new way to disable and ultimately eliminate the HBV cccDNA and prevent its expression. Our hypothesis is that incorporation of HMPMA into the DNA induces structural abnormalities, and in this case possibly destabilizing cccDNA, altering its structure and leading to its elimination over time. Our team combines the state of the art medicinal chemistry (Hostetler) and HBV molecular biology (Siddiqui) expertise to address this long standing problem of HBV viral persistence of cccDNA in chronic infection and being refractory to antiviral strategies currently in vogue.